The quantitation of RNA, particularly RNA derived from infectious agents or from cellular sources, is important in the diagnosis and monitoring of disease states caused by such agents. For example, the viral load detected in serum correlates to high concentrations of virus in the lymph nodes and has predictive value in assessing progression of AIDS to advanced stages, as reported in Ho, et al., Nature, 373: 123 (1995) and Mellors, et al., Ann. Intern. Med., 122: 573 (1996). Viral titers in serum are also correlated with disease progression for other viruses such as HCV, nonA nonB hepatitis other than HCV, and atypical lentiviruses.
There are several fundamental problems in RNA quantitation in low copy number. If there are too few molecules to detect by conventional means, amplification of the target sequence to increase its numbers by several logs is necessary. However, the coefficients of variability (CV) may often exceed 20 percent or more, so that the result obtained is unreliable, and does not correlate with the stage of disease. The coefficient of variability (CV) is defined as the standard deviation of the values obtained divided by the mean. Increased signal from a small number of target molecules is another approach, but the final result depends upon a large number of reactions which must occur in correct sequence. Again, there is a large CV because of reaction sequence errors giving a spectrum of values.
Alternatively, direct measurement of the low RNA copy number in the native sample, even where adequate detection sensitivities can be achieved, is thwarted by the inherent instability of RNA-DNA duplexes. Increasing the length of the hybridized target can increase both sensitivity and stability of the hybrid, but the additional nucleotide sequence combinations increase the chance of nonspecific hybridizing to fragments of host nucleic acids or partial hybridization to nonselected regions of the viral genome, thereby contributing to a falsely inflated positive value. Most of the improvements to date in low RNA copy number quantitation represent attempts to better control the multiple molecular events involved in signal or nucleic acid amplification strategies.
The three main amplification systems currently available include branched chain signal amplification (bDNA), reverse transcriptase polymerase chain reaction (RT-PCR), and nucleic acid sequence based amplification (NASBA). The strategy of the first two, bDNA and RT-PCR, involves using a first reaction step that converts the system from an RNA target to a DNA target.
In bDNA an initial probe hybridizing with a complementary probe contains a plurality of noncomplementary sites capable of hybridizing to further DNA strands, which in turn may hybridize sites noncomplementary to the probe sequence, so that as repeated layers of hybridization occur, a branched DNA structure of extreme complexity is created. The last to be annealed strand in the branched structure carries a reporter. Thus the original RNA target molecule gives rise to an amplification of the signal generating capability of the system. A full explanation and description of the bDNA technique is set forth in Fultz, et al., "Quantitation of plasma HIV-I RNA using an ultra sensitive branched DNA (bDNA) assay", in Program and Abstracts of the 2nd National Conference on Human Retroviruses (1995), and product literature, L-6170 Rev. 5.0 for the Quantiplex.TM. HIV-RNA Assay (Chiron Corporation).
In RT-PCR a cDNA is generated from the RNA template, and then an ordinary PCR amplification ensues utilizing selected primers to define the left and right ends of the amplicon. Each successive round of synthesis and denaturation causes an exponential increase in the number of progeny strands generated in the system. After the amplification is complete, a probe having a complementary sequence to some portion of the amplicon and carrying a reporter can be used for detecting the amplified target. In both RT-PCR and bDNA, the original RNA target can theoretically be dispensed with, without impairing the sensitivity of the test, once the conversion to a DNA system has occurred. These methods effectively get around the inherent lability of the RNA target or its RNA-DNA duplex hybrid.
Similarly, both RT-PCR and bDNA share many of the same deficiencies. Both systems rely upon the integrity of a large number of successive hybridization events. If an early hybridization event fails, for any of a number of reasons such as structural (steric) hindrance, uncorrected mismatch, binding of a defective enzyme molecule, etc., the final number of copies, and therefore the intensity of the signal will be ablated. These random occurrences help to account for the great sensitivity of the assays coupled with a widely variable coefficient of variability. The commercial form of the test normalizes variability by co-amplification of an internal standard. To control for variability the internal standard must be amplified under identical conditions as the target yet be able to be differentiated from the target, an almost impossible task. Also, introducing an internal standard changes the PCR reaction kinetics itself. RT-PCR, while showing some efficacy, is in practice very labor intensive, and not practical under normal clinical laboratory conditions.
In NASBA, the lability of RNA is overcome by increasing copy number to a vast number. The technique involves creating a cDNA from the target RNA and then generating a large number of transcripts from bDNA template, which in turn can be converted to a cDNA, and so on. The number of transcripts produced is always much greater than the number converted to cDNA, so that a large excess of RNA occurs. The process is initiated upon annealing of two primers, one of which contains a phage promoter, which in the ensuing cDNA provides a point of initiation for transcription. Unlike PCR where the numbers of actual cycles of amplification are nominally controlled by the number of temperature cycles, there is much less control in NASBA. The technique suffers from a lack of uniformity as between different target sequences, and in the same target sequence from one run to another. The commercial form of the assay employs three internal calibrators, which are co-amplified with the target sequence. In any detection technique it is desirable for the analytical coefficient of variability (CV) to be less than 15 percent.
The three techniques were recently compared in a study by Coste, et al., J. Med. Virol., 50: 293 (1996). bDNA was found to be most reproducible with CVs ranging from 6-35 percent. Better results were achieved at high copy number, 12.4% vs. 31% for low copy number. However, sensitivity was only 68 percent with a lower level of detection at 4000 HIV equivalents. NASBA was the least reliable test with CVs ranging from 13-62 percent, with CV averages of 20.7 percent for high copy number and 41.8 percent for low copy number. Sensitivity was 100 percent with a lower level of detection at 2600 HIV equivalents. Finally, RT-PCR has a sensitivity of 93 percent, but a mean CV of 43 percent.
While improvements in the foregoing techniques may result from optimization of the operating conditions of the assays, and from discovery of reagent combinations that minimize interferences with hybridizations, it is unlikely that variability will ever be reduced uniformly to coefficient values less than 15 percent. This is because priming errors and hybridization interferences cannot be entirely overcome, and misevents occurring early in the sequence of amplification steps have a geometric impact on the result. Thus the wide range of CV. If the level of sensitivity for direct detection of RNA could be increased by several orders of magnitude over standard UV detection methods, and the problem of RNA-DNA duplex instability be solved, direct detection would provide a viable alternative to current amplification-based methods without loss of reliability.